Novel envelope protein time-resolved fluoroimmunoassay as an alternative in vitro potency assay for quality control of inactivated Japanese encephalitis virus vaccine.

Japanese encephalitis (JE) vaccination is the most effective way to prevent JE. Plaque reduction neutralization test (PRNT) as the standard method for potency testing for inactivated JE vaccine could not provide the exact potency value. Envelope (E) protein of JE virus induces the body to create neutralizing antibodies. There is a potential for using the determination of E protein to assess the immunogenicity and efficacy of JE vaccine. In this study, an automatic time-resolved fluoroimmunoassay for detection of E protein in JE vaccine was established as a simple and rapid in vitro potency assay to complement PRNT, including the expression and paired screening of monoclonal antibodies, the establishment of assay method and performance verification. A pair of anti-E protein neutralizing antibodies (L022 and L034) were screened to construct the sandwich detection pattern. After pre-treating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a little detection time and eliminated manual error. The results of the validation experiment met the requirements for quality control. The linear range was from 0.78125 U/mL to 25 U/mL, the sensitivity was 0.01 U/mL, the intra-assay coefficient of variation was less than 5 %, and the inter-assay coefficient of variation was less than 10 %. The recovery from the dilution was between 90 % and 110 %. This present TRFIA shown good stability and effectiveness in quality control for samples related to JE vaccine production. The outcomes demonstrated that the present TRFIA could be an alternative in vitro potency assay in quality control for inactivated JE vaccine.

A time-resolved fluorescence immunoassay for rapid and precise automatic quality control of human papillomavirus type 68 VLPs in human papillomavirus vaccine.

The effectiveness and necessity of human papillomavirus (HPV) vaccination to prevent HPV infection and cervical cancer are increasingly recognized by people. The 15-valent HPV vaccine, which protects against almost high-risk types of HPV viruses identified by WHO, has attracted much attention. However, as the valence of vaccines increases, quality control in the HPV vaccine production process is facing more challenges. The precise quality control of the HPV type 68 virus-like particles (VLPs), one of the unique components of the 15-valent HPV vaccine that distinguishes it from existing vaccines, is the new requirement for vaccine manufacturers. Here we developed a novel time-resolved fluorescence immunoassay (TRFIA) for rapid and precise automatic quality control of HPV68 VLPs in HPV vaccine. Two murine monoclonal antibodies specifically targeting the HPV68 L1 protein were used to establish a classical sandwich assay. Except for pretreating the vaccine sample, the whole analysis process was performed by a fully automated machine, which saves detection time and gets rid of manual error. Multiple experiments established that the current novel TRFIA can efficiently and reliably analyses HPV68 VLPs. Present novel TRFIA has exhibited merits with speed, robustness, high sensitivity with a minimum detection value of 0.08 ng/mL, considerable accuracy, a wide detection range (up to 1000 ng/mL) and excellent specificity. It is also expected to provide a new detection method for quality control for each HPV type VLPs. To summarize, the novel TRFIA is of great interest for application in HPV vaccine quality control.

A custom-made time-resolved fluoroimmunoassay for the quantitation of the host cell protein of Vero in rabies vaccine.

Host cell proteins (HCPs) are the process-specific and inevitable impurities during the manufacture via a host cell, which affect the safety or efficacy of the bio-product. However, the commercial HCP enzyme-linked immunosorbent assay (ELISA) kits may not apply to specific products such as rabies vaccine from Vero cells. More advanced and process-specific assay methods are needed in the quality control of rabies vaccine throughout the whole manufacturing process. Therefore, a novel time-resolved fluoroimmunoassay (TRFIA) for the detection of process-specific HCP of Vero cells in rabies vaccine was established in this study. Liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) was used during the preparation of HCP antigen. Based on a sandwich-type immunoassay format, analytes in samples were captured by one antibody coating in the wells and "sandwiched" by another antibody labeled with europium chelates. Due to the complex composition of HCP, both the capture and detected antibodies are polyclonal antibodies from the same anti-HCP antibodies pool. Multiple experiments have identified the optimal conditions to allow the valid and reliable detection of HCP in rabies vaccine. The TRFIA had a satisfactory limit of detection value (0.011 µg/ml) under optimal conditions, with the linear range from 0.0375 to 2.4 µg/ml of HCP. The coefficient variations (CVs) were all < 10%, and the recoveries were in the range of 97.00-102.42%. All the test results of Vero cell protein reference substance were included in the expected concentration, which demonstrated that the present method was available for the test of HCP in rabies vaccine. Based on these results, the novel TRFIA to detect HCP appears to be important for application in modern vaccine quality control during the whole manufacturing process.

Colonoscopy-induced acute appendicitis: A case report.

BACKGROUND: Colonoscopy is widely used for examination, diagnosis, and treatment because of its low incidence of associated complications. Post-colonoscopy appendicitis (PCA) is very rare and is easily misdiagnosed as electrocoagulation syndrome or colon perforation. Therefore, clinicians should pay close attention to this complication. CASE SUMMARY: A 47-year-old female patient underwent a colonoscopy for a systematic physical examination, and the procedure was uneventful with normal endoscopic and histologic findings. However, the bowel preparation was suboptimal (Boston 2-3-2). After the examination, the patient experienced pain in the lower abdomen, which progressively worsened. Computed tomography of the lower abdomen and pelvis revealed appendiceal calcular obstruction and appendicitis. As the patient refused surgery, she was managed with antibiotics and recovered well. CONCLUSION: In the current literature, the definition of PCA remains unclear. However, abdominal pain after colonoscopy should be differentiated from acute appendicitis.

Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways.

A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

Europium (III) chelate nanoparticle-based lateral flow immunoassay strips for rapid and quantitative detection of cystatin C in serum.

The concentration of Cys C in the patient's serum can reflect the level of glomerular filtration rate and indicate the occurrence of renal failure. The establishment of a simple and rapid analytical method to quantitatively monitor the concentration of Cys C in serum could help timely detection of renal failure. In this study, we have developed an Eu (III) chelate nanoparticles based lateral flow immunoassay to fulfill real-time monitoring of Cys C concentration in serum within 15 min. This method was performed as a sandwich immunoassay with a wide detection range (0.05-10 µg/mL) and a low limit of detection (24.54 ng/mL). The intra and inter-assay coefficients of variation were 8.31-8.61% and 8.92-9.95%, respectively. Furthermore, the application of this method was evaluated by comparing the determined results with those obtained by chemiluminescence immunoassay, exhibiting a satisfactory correlation (R2 = 0.9830). The developed LFIA method with satisfactory analytical performance has great potential for real-time monitoring of renal failure and self-detection for the high-risk population.

Discovery of SHR5133, a Highly Potent and Novel HBV Capsid Assembly Modulator.

Capsid assembly modulators (CpAMs) represent a new class of antivirals targeting hepatitis B virus (HBV) core protein to disrupt the assembly process. In this work, a novel chemotype featuring a fused heterocycle amide was discovered through pharmacophore exploration. Lead optimization resulted in compound 8 with an EC50 value of 511 nM, and then methyl substitution on the piperazine was found to improve the in vitro potency remarkably. Further SAR studies established the key compound SHR5133, which showed high in vitro antiviral potency, favorable pharmacokinetic profiles across species, and robust in vivo efficacy.

Head-to-head comparison of 7 high-sensitive human papillomavirus nucleic acid detection technologies with the SPF10 LiPA-25 system.

Background: The SPF10 LiPA-25 system for human papillomavirus (HPV) detection with high analytical performance is widely used in HPV vaccine clinical trials. To develop and evaluate more valent HPV vaccines, other comparable methods with simpler operations are needed. Methods: The performance of the LiPA-25 against that of other 7 assays, including 4 systems based on reverse hybridization (Bohui-24, Yaneng-23, Tellgen-27, and Hybribio-16) and 3 real-time polymerase chain reaction (PCR) assays (Hybribio-23, Bioperfectus-21, and Sansure-26), was evaluated in selected 1726 cervical swab and 56 biopsy samples. A total of 15 HPV genotypes (HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66) were considered for comparison for each HPV type. Results: Among the swab samples, compared to LiPA-25, compatible genotypes were observed in 94.1% of samples for Hybribio-23, 92.8% for Yaneng-23, 92.6% for Bioperfectus-21, 92.4% for Hybribio-16, 91.3% for Sansure-26, 89.7% for Bohui-24, and 88.0% for Tellgen-27. The highest overall agreement of the 15 HPV genotypes combined was noted for Hybribio-23 (κ = 0.879, McNemar's test: P = 0.136), followed closely by Hybribio-16 (κ = 0.877, P< 0.001), Yaneng-23 (κ = 0.871, P < 0.001), Bioperfectus-21 (κ = 0.848, P < 0.001), Bohui-24 (κ = 0.847, P < 0.001), Tellgen-27 (κ = 0.831, P < 0.001), and Sansure-26 (κ = 0.826, P < 0.001). Additionally, these systems were also highly consistent with LiPA-25 for biopsy specimens (all, κ > 0.897). Conclusions: The levels of agreement for the detection of 15 HPV types between other 7 assays and LiPA-25 were all good, and Hybribio-23 was most comparable to LiPA-25. The testing operation of HPV genotyping should also be considered for vaccine and epidemiological studies.

A chemiluminescence immunoassay for precise automatic quality control of glycoprotein in human rabies vaccine.

Currently, quality control of glycoprotein in the human rabies vaccine is based on enzyme-linked immunosorbent assay (ELISA). However, ELISA does not match the needs of a modernised quality control system. For a long time, human rabies virus vaccine manufacturers have been devoted to seeking a detection platform that is sensitive, accurate, automatic, and feasible for practical applications. Therefore, our team invested major efforts into establishing a fully automated micromagnetic particle (MMP)-based chemiluminescence immunoassay (CLIA) platform. For vaccine quality control, MMP-coupled rabies virus glycoprotein monoclonal antibodies (S037) were used to capture the rabies virus. Another rabies virus glycoprotein antibody (S053) labelled with acridinium ester was added as a signal tracer. After pretreating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a limited detection time (only 30 min) and eliminated manual error. Multiple experiments have identified the optimal conditions allowing valid and reliable assessment of vaccine potency. The CLIA platform has exhibited merits in terms of speed, robustness, high sensitivity (with a minimum detection value of 0.45 mIU/mL), considerable accuracy, and a wide linear range of detection (9.4-1200 mIU/mL). Furthermore, the results showed that the CLIA platform is consistent with the National Institutes of Health test and time-resolved fluorescent immunoassay (TRFIA) in quantitative analysis, and had a better analytic performance than TRFIA. Therefore, the CLIA platform presented here may be important for application in modern vaccine quality control.

CRISPR-Cas13a-based diagnostic method for Chlamydia trachomatis from nongonococcal urethritis.

Aim: Development of a routine screening technique for Chlamydia trachomatis infection. The proposed approach involves the CRISPR RNA (crRNA). In the presence of the target sequence, the RNase activity of the Cas13a protein is activated, and it cleaves RNA fluorescent probe so that fluorescence will be emitted. Results: The sensitivity of the detection based on CRISPR-Cas13a was 10 fM. The results obtained by CRISPR-Cas13a and quantitative polymerase chain reaction were closely correlated: χ2 = 81.798 (p < 0.001). Conclusion: The method can be carried out at room temperature and yields results within 2 h. The developed technique does not require expensive instruments and, thus, can meet the needs of community hospitals and other institutions for screening.

Rapid and Sensitive Detection of anti-SARS-CoV-2 IgG, Using Lanthanide-Doped Nanoparticles-Based Lateral Flow Immunoassay.

The outbreak of 2019 coronavirus disease (COVID-19) has been a challenge for hospital laboratories because of the huge number of samples that must be tested for the presence of the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Simple and rapid immunodiagnostic methods are urgently needed to identify positive cases. Here we report the development of a rapid and sensitive lateral flow immunoassay (LFIA) that uses lanthanide-doped polysterene nanoparticles (LNPs) to detect anti-SARV-CoV-2 IgG in human serum. A recombinant nucleocapsid phosphoprotein of SARS-CoV-2 was dispensed onto a nitrocellulose membrane to capture specific IgG. Mouse anti-human IgG antibody was labeled with self-assembled LNPs that served as a fluorescent reporter. A 100-µL aliquot of serum samples (1:1000 dilution) was used for this assay and the whole detection process took 10 min. The results of the validation experiment met the requirements for clinical diagnostic reagents. A value of 0.0666 was defined as the cutoff value by assaying 51 normal samples. We tested 7 samples that were positive by reverse-transcription (RT-)PCR and 12 that were negative but clinically suspicious for the presence of anti-SARS-CoV-2 IgG. One of the negative samples was determined to be SARS-CoV-2 IgG positive, while the results for the other samples were consistent with those obtained by RT-PCR. Thus, this assay can achieve rapid and sensitive detection of anti-SARS-CoV-2 IgG in human serum and allow positive identification in suspicious cases; it can also be useful for monitoring the progression COVID-19 and evaluating patients' response to treatment.

Discovery of SHR1653, a Highly Potent and Selective OTR Antagonist with Improved Blood-Brain Barrier Penetration.

The oxytocin receptor (OTR) plays a major role in the control of male sexual responses. Antagonists of the OTR have been reported to inhibit ejaculation in animal models and serve as a potential treatment for premature ejaculation (PE). Herein, we describe a novel scaffold featuring an aryl substituted 3-azabicyclo [3.1.0] hexane structure. The lead compound, SHR1653, was shown to be a highly potent OTR antagonist, which exhibited excellent selectivity over V1AR, V1BR, and V2R. This novel molecule was shown to have a favorable pharmacokinetic profile across species, as well as robust in vivo efficacy in a rat uterine contraction model. Interestingly, SHR1653 exhibited excellent blood-brain barrier penetration, which might be beneficial for the treatment of CNS-related PE.

Ultrasensitive Sensor Using Quantum Dots-Doped Polystyrene Nanospheres for Clinical Diagnostics of Low-Volume Serum Samples.

Development of sensitive homogeneous assays is a high-priority research target for clinical diagnostics. Quantum dots (QDs) present favorable photophysical properties, which implies their potential as an exceptional dye in fluorescence detection. QDs-based biosensors have been described in the literature; however, few of them have truly progressed to widespread clinical usage. In this work, a chemiluminescent homogeneous detecting biosensor is fabricated using QDs-doped polystyrene nanospheres to sensitively detect biomarkers in low-volume serum samples. Phthalocyanine-dyed and QDs-encapsulated carboxylate-functionalized polystyrene nanospheres with surface carboxyl groups (PPs and QPs, respectively) were fabricated and served as triggers and fluorescent probes, respectively, in this biosensing system. In this sandwich-format immunoassay, the PPs produced singlet oxygen once sensitized by 680 nm diode lasers, and the QPs, conjugated with antibodies, and then reacted with the singlet oxygen in the presence of specific antigens and emitted anti-Stokes fluorescence with wavelengths around 605 nm, as a result of fluorescence resonance energy transfer (FRET) within the QPs. We demonstrated the determination of carcinoembryonic antigen as a model protein target in 25 µL of serum samples with an unprecedented detection limit of 2.56 × 10-13 M (46 pg/mL) using this biosensor. Furthermore, excellent correlations ( R2 = 0.99718, n = 107) were obtained between utilizing this biosensor and commercialized chemiluminescence immunoassay kits in clinical serum detection. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be a promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for patients.

A Scalable Total Synthesis of the Antitumor Agents Et-743 and Lurbinectedin.

An efficient and scalable approach is described for the total synthesis of the marine natural product Et-743 and its derivative lubinectedin, which are valuable antitumor compounds. The method delivers 1.6 % overall yield in 26 total steps from Cbz-protected (S)-tyrosine. It features the use of a common advanced intermediate to create the right and left parts of these compounds, and a light-mediated remote C-H bond activation to assemble a benzo[1,3]dioxole-containing intermediate.

NLRP3 inflammasomes in macrophages drive colorectal cancer metastasis to the liver.

Inflammation and immunity are important determinants of cancer initiation, promotion, and progression to cancer equilibrium or suppression. The NOD-like receptor family pyrin domain containing 3 (NLRP3) is an oligomeric intracellular immune receptor, and the main component of inflammasome. As a widely distributed effector of innate immunity, NLRP3 inflammasome affects development of many cancer types, but its exact role in colorectal cancer (CRC) is controversial. We found that cells with the macrophage (MΦ) marker CD68 and strong NLRP3 expression densely surrounded CRC tissue. The NLRP3 inflammasome was activated in MΦs by MΦ-CRC cell crosstalk; it resulted in faster migration of CRC cells, whereas blocking NLRP3 signaling suppressed CRC cell migration in vitro, and metastatic ability in vivo. NLRP3 signaling activation in MΦs can contribute to CRC cell migration and invasion.

Translationally controlled tumor protein affects colorectal cancer metastasis through the high mobility group box 1-dependent pathway.

Recently, accumulating evidence from clinical and experimental researches have suggested that translationally controlled tumor protein (TCTP) and high mobility group box 1 (HMGB1) are implicated in colorectal cancer (CRC) metastasis. However, whether there is an interconnection between these two tumor-promoting proteins and how they affect CRC metastasis remain to be fully elucidated. In the present study, the expression level of TCTP in CRC tissues was assessed by immunohistochemical staining and immunoblotting, and the serum concentration of HMGB1 in patients with CRC was detected by enzyme-linked immunosorbent assay. In vitro, following the modulation of TCTP expression in colon cancer LoVo cells, the translocation behavior of HMGB1 was observed by immunofluorescence assay. Furthermore, the activity of nuclear factor-κB (NF-κB) in LoVo cells was evaluated by immunoblotting and luciferase assay, and the invasion ability of LoVo cells after different treatments was determined using cell invasion assay. In vivo, xenograft tumor model was established and the correlation of TCTP and HMGB1 expression in xenografted tumors was studied by immunohistochemical examination. The results revealed that the expression level of TCTP in CRC tissue and the serum concentration of HMGB1 in patients with CRC were significantly increased, and there was a strong positive correlation between them. In vitro experiments showed that the overexpression of TCTP on LoVo cells resulted in the release of HMGB1 from the nucleus to the cytoplasm and into the extracellular space. In addition, the overexpression of TCTP led to the activation of NF-κB in LoVo cells, and this effect was reversed by treatment with antibodies targeting HMGB1 or to its receptors Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products advanced glycation end products (RAGE). Furthermore, inhibition of the HMGB1-TLR4/RAGE-NF-κB pathway significantly inhibited the TCTP-stimulated invasion of LoVo cells. In vivo experiments demonstrated that the overexpression of TCTP in nude mice promoted the development and spread of xenografted tumors, and concurrently enhanced the expression of HMGB1 in tumor tissues. Collectively, these findings suggested that TCTP promotes CRC metastasis through regulating the behaviors of HMGB1 and the downstream activation of the NF-κB signaling pathway.

Oncogene mutational analysis in Chinese gastrointestinal stromal tumor patients.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors and exhibit a high frequency of oncogenic KIT or PDGFRA mutations. Tyrosine kinase inhibitors (TKIs) have been mainly used in the treatment of GISTs bearing KIT/PDGFRA mutations. However, other mutation profiles have been found to affect the sensitivity to and effectiveness of TKIs in the treatment of GISTs. PURPOSE: The aim of the present study was to describe the mutational status of multiple genes in GIST samples and to provide information for finding potential predictive markers of therapeutic targets in Chinese GIST patients. PATIENTS AND METHODS: MassARRAY spectrometry was used to test 40 Chinese GIST patients for 238 mutations affecting 19 oncogenes. RESULTS: A total of 14 oncogenes with 43 mutations were detected in 38 samples, with a mutation frequency of 95%. Among these mutation samples, 26 GISTs were found for KIT or PDGFRA mutations, while 12 were KIT/PDGFRA wild-type. Approximately half of the GIST samples harbored multiple mutations. The most frequent mutations were found in KIT (62.5%), CDK4 (17.5%), NRAS (15%) and EGFR (12.5%). Other mutations included PIK3CA and AKT1 (10%), BRAF and ABL1 (7.5%), PDGFRA, ERBB2 and HRAS (5%), and AKT2, FLT3 and KRAS (2.5%). New mutated genes (CDK4, AKT2, FLT3, ERBB2, ABL1 and AKT1), a higher BRAF mutation frequency (7.5%) and new BRAF mutation sites (G464E) were found in Chinese GIST patients. CONCLUSION: This study demonstrated useful mutations in a small fraction of Chinese GIST, but targeted therapeutics on these potential predictive markers need to be investigated in depth especially in Oriental populations.

Simple and rapid monitoring of doxorubicin using streptavidin-modified microparticle-based time-resolved fluorescence immunoassay.

Developing a simple analytical method suitable for therapeutic drug monitoring in a clinical setting is key to establishing guidelines on accurate dose administration and the advancement of precision medicine. We devised a simple rapid analytical method through the combination of streptavidin-modified microparticles and a time-resolved fluorescence immunoassay for therapeutic drug monitoring. The analytical performance of this method was investigated and validated using clinical samples. By determination of doxorubicin concentration, the proposed assay has shown a satisfactory linear range of detection (3.8-3000 ng mL-1) with a limit of detection of 3.8 ng mL-1 and an IC50 of 903.9 ng mL-1. The intra and inter-assay coefficients of variation were 4.12-5.72% and 5.48-6.91%, respectively, and the recovery was acceptable. The applicability of the proposed assay was assessed by comparing the determined results with those measured by LC-MS/MS, presenting a satisfactory correlation (R 2 = 0.9868). The proposed assay, which shows satisfactory analytical performance, has great potential for application in the field of TDM in the future.

Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S, 25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals Non-Identity with the Natural Product.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki-Hiyama-Kishi coupling to form the C21-C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

Xanthine Oxidase Induces Foam Cell Formation through LOX-1 and NLRP3 Activation.

PURPOSE: Xanthine oxidase catalyzes the oxidation of xanthine to uric acid. This process generates excessive reactive oxygen species (ROS) that play an important role in atherogenesis. Recent studies show that LRR and PYD domains-containing protein 3 (NLRP3), a component of the inflammasome, may be involved in the formation of foam cells, a hallmark of atherosclerosis. This study was designed to study the role of various scavenger receptors and NLRP3 inflammasome in xanthine oxidase and uric acid-induced foam cell formation. METHODS AND RESULTS: Human vascular smooth muscle cells (VSMCs) and THP-1 macrophages were treated with xanthine oxidase or uric acid. Xanthine oxidase treatment (of both VSMCs and THP-1 cells) resulted in foam cell formation in concert with generation of ROS and expression of cluster of differentiation 36 (CD36) and oxidized low density lipoprotein (lectin-like) receptor 1 (LOX-1), but not of scavenger receptor A (SRA). Uric acid treatment resulted in foam cell formation, ROS generation and expression of CD36, but not of LOX-1 or SRA. Further, treatment of cells with xanthine oxidase, but not uric acid, activated NLRP3 and its downstream pro-inflammatory signals- caspase-1, interleukin (IL)-1ß and IL-18. Blockade of LOX-1 or NLRP3 inflammasome with specific siRNAs reduced xanthine oxidase-induced foam cell formation, ROS generation and activation of NLRP3 and downstream signals. CONCLUSIONS: Xanthine oxidase induces foam cell formation in large part through activation of LOX-1 - NLRP3 pathway in both VSMCs and THP-1 cells, but uric acid-induced foam cell formation is exclusively through CD36 pathway. Further, LOX-1 activation is upstream of NLRP3 activation. Graphical Abstract Steps in the formation of foam cells in response to xanthine oxidase and uric acid. Xanthine oxidase stimulates LOX-1 expression on the cell membrane of macrophages and vascular smooth muscle cells (VSMCs) and increases generation of ROS, which activate NLRP3 inflammasome and downstream pro-inflammatory mediators such as Caspase-1, IL-1ß and IL-18. Xanthine oxidase also induces CD36 expression. Activation of both LOX-1 and CD36 (LOX-1> > CD36) participates in the transformation of macrophages and VSMCs into foam cells. Uric acid formed from xanthine-xanthine oxidase interaction stimulates CD36 expression and triggers foam cell formation independent of NLRP3 activation.